Through the use of natural template probes many components of the E. coli replicative machinery have been isolated. These components include a priming enzyme, a single-stranded DNA binding protein and the complex DNA polymerase III holoenzyme that consists of at least seven different subunits. The functional role of many replication proteins is becoming understood; however, little is known of the regulation and coordination of their synthesis. This project proposes to investigate this regulatory problem by using recombinant plasmids (i) to increase in vivo the concentration of a specific replication gene and (ii) to direct an in vitro protein synthesizing system. The effect of gene dosage upon other replication gene products will be examined by functional and immunological assays. This may detect coordinate relationships between replication protein levels. An in vitro protein synthesizing system may (i) permit identification of the protein product of a given dna gene, (ii) allow detection of precursors to active replication proteins, and (iii) provide an assay for specific effectors of replication protein transcription or translation.